distribution of moulds and mycotoxins in maize...

Biotechnology in Animal Husbandry 33 (3), p 309-319 , 2017 ISSN 1450-9156

Publisher: Institute for Animal Husbandry, Belgrade-Zemun UDC 614.91'639

https://doi.org/10.2298/BAH1703309Y

PROTEINS SEQUENCE ANALYSIS OF CONTAGIOUS

CAPRINE PLEUROPNEUMONIA

Ayuba Dauda 1,Abdulmojeed Yakubu2, Ihe Ndu Dim1, Deeve Sebastian

Gwaza 1

1University of Agriculture Makurdi, P. M. B 2373 Makurdi, Nigeria 2State University Lafia Campus Nasarawa State, Nigeria

Corresponding author: [email protected]

Abstract. A total of twenty (20) contagious bovine pleuropneumonia

(CCPP) proteins were retrieved from the GenBank (www.ncbi.nlm.nih.gov). The

proteins sequences were used to investigate the molecular identity of various CCPP

proteins. The physico-chemical properties of CCPP proteins were performed using

protparam tool. Isoelectric point (pI), molecular weight (MW), extinction

coefficient (EC); instability index (II), aliphatic index (AI) and grand average of

hydropathicity (GRAVY) were computed. The study revealed that the pI of CCPP

proteins were acidic and basic in nature. The EC and II of CCPP proteins indicate

better stability which is an indication of resistant to mutation and thermally stable.

The GRAVY of CCPP proteins revealed some are positive while some are

negative. The positive value indicates solubility (hydrophilic) in water while

negative is not soluble (hydrophobic) in water. The amino acid composition of

CCPP proteins indicates that they are rich in isoleucine, leucine and lysine. The

three dimensional structures (3D) of the CCPP proteins were determine using

Phyre2 server. The amino acid sequences of CCPP proteins were subjected to

secondary structure prediction using ExPASy’s SOPMA tool. The proteins are

more of alpha helix structure. The genetic information eminating from this study

may bring insight into mutagenesis and pharmacogenetic.

Keywords: protein, caprine pleuropneumonia, Sequence

https://doi.org/10.2298/BAH1803369E

Introduction

Contagious Caprine Pleuropneumonia (CCPP) is a devastating disease of

goats included in the list of notifiable diseases of the Organization for Animal

Health (OIE).The first description of the disease dates back to 1873, in Algeria

(Thomas, 1873).CCPP is a contagious disease of goats, which occurs in per acute,

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acute or chronic forms and is characterized by fibrinous pneumonia, pleurisy and

profuse pleural exudates (Edelsten et al., 1990). Mortality rates of 60–100% are

common (Edelsten et al., 1990). The disease is reported to occur in many countries

in West and Eastern Africa and in Pakistan and India (OIE, 2001). The infectious

agent Mycoplasma capricoleum subspecies capripneumoniae, formerly known as

the F38-like group, is difficult to isolate and has only been identified in a few of

the countries where the disease has been reported (Bolske et al., 1995a).

Materials and Methods

A total of twenty (20) CCPP proteins of goat were retrieved from the

GenBank (www.ncbi.nlm.nih.gov). The Genbank accession numbers of the

sequences and sequence variations are shown in Table 1. ProtParam Tool was used

for the computation of various physical and chemical properties of the CCPP

proteins using amino acid sequences. The computated parameters were molecular

weight, theoretical pI (isoelectric point), amino acid composition, extinction

coefficient, estimated half-life, instability index, aliphatic index and grand average

of hydropathicity (GRAVY) (Gasteiger, 2005). The amino acid sequences of

CCPP proteins were subjected to secondary structure prediction using ExPASy’s

SOPMA tool. It predicts 69.5% of amino acids for a 3 state description of the

secondary structure (a helix, b sheets and coil). The Phyre2 server was used to

predict the 3D structure of CCPP proteins. These servers predict the three-

dimensional structure of a protein sequence using the principles and techniques of

homology modeling (Kelley and Sternberg, 2009). Currently, the most powerful

and accurate methods for detecting and aligning remotely related sequences rely on

profiles or Hidden Markov Models (HMMs). 3DligandSite was used to predict the

binding site of the 3D structure of the CCPP proteins. Phyre2 is coupled to the

3DligandSite server for protein binding site prediction (Wass et al., 2010).

Results

Physico-chemical characteristics of CCPP proteins predicted by protparam are shown in Table 2. The computed isoelectric point (pI) values of CCPP proteins in the study revealed Phosphoglycerate kinase, Glycyl-tRNA Synthetase, ATP-dependent protease La, GTP-Binding protein, tRNA Modification GTpass, Lysine-tRNA ligase and Chromosome segregation ATPase are acidic which have (pI7). The net charge of CCPP protein revealed only Phosphoglycerate kinase is neutral (no charge). Glycyl-tRNA Synthetase, ATP-dependent protease La, GTP-Binding protein, tRNA

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Modification, Lysine-tRNA ligase and Chromosome segregation ATPase are negatively (-) charge while the rest of the protein are positively (+) charge. The extinction coefficient of a protein at 280 nm depends almost exclusively on the number of aromatic residues, particularly tryptophan (Gill et al., 1989). Extinction coefficient values for CCPP proteins at 280 nm ranged from 8940 to 143950

(Signal recognition particle protein is lowest and Prolipoprotein diacylglyceryl tranferase is highest) respectively. Table 1: Protein name, accession, and number of goat CCPP

S/N NAME OF PROTEIN ACCESSION No AMINO ACID No

1 Phosphoglycerate kinase KEY8461 404

2 Chaperone protein Dnaj KEY84219 372

3 Amino acid permease KEY84758 515

4 Glycyl-tRNA Synthetase KEY84567 456

5 GTP pyrophosphokinase KEY84560 754

6 ATP-dependent protease La KEY84622 779

7 DNA-primase KEY84568 604

8 Histidy-tRNA Synthetase KEY84179 414

9 GTP-Binding protein KEY84763 364

10 Excinuclease ABC Subunit B KEY84755 665

11 tRNA Modification GTpass KEY84779 452

12 Tyrosine-TRNA ligase KEY84654 414

13 PTS system-IIBC component KEY84580 602

14 Hypothetical Protein Mccp 3340 KEY84577 820

15 Dihydro folate-foly poly glutamate synthase

KEY84753 369

16 Lysine-tRNA ligase KEY84440 500

17 Prolipoprotein diacylglyceryl tranferase

KEY84751 526

18 Chromosome segregation ATPase KEY84561 988

19 Cell division protein FtsY KEY84539 424

20 Signal recognition particle protein KEY84596 447

The half life of protein is the time it takes for half of the amount of protein

in a cell to disappear after its synthesis in the cell of the proteins. In this study the half life of all the CCPP proteins is 30 hours.The instability index provides an estimate of the stability of the protein in a test tube. A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein will be unstable (Guruprasad et al., 1990). The result from this study shown that ATP-dependent protease La, Excinuclease ABC Subunit B, Prolipoprotein diacylglyceryl tranferase and Cell division protein FtsY protein have value >40 while the rest of protein are have

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IIBC component, Hypothetical Protein Mccp 3340, Dihydro folate-foly poly glutamate synthase and Prolipoprotein diacylglyceryl tranferase proteins from this study have AI>100 while the rest of the CCPP protein have AI


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highest value (28.18%) and the lowest is Signal recognition particle protein (13.87%). The beta turn prediction of secondary structure revealed that Chaperone protein Dnaj gives the highest value (14.25%) and Chromosome segregation ATPase is the lowest (5.67%). The random coil prediction of secondary structure revealed that Chaperone protein Dnaj gives the highest value (35.22%) and Chromosome segregation ATPase showed the lowest value (20.04%). All the CCPP proteins are having higher value in alpha helix structure. The amino acid composition percentage of CCPP protein is shown in Table 4. All the CCPP proteins used for this study have similar amino acid composition of all the CCPP protein with higher percentage in isoleucine, leucine and lysine. Isoleucine and leucine are aliphatic amino acid and lysine is polar amino amino acid. All the CCPP proteins have zero percentage composition of selenocystein and pyrrolysine amino acids.

Table 3: Prediction of secondary structures of CCPP proteins

Protein Alpha

Helix (%)

Extended Strand (%) Beta Turn

(%)

Random

Coil (%)

Phosphoglycerate kinase 42.57 21.78 12.62 23.02

Chaperone protein Dnaj 26.88 23.66 14.25 35.22

Amino acid permease 39.03 27.57 10.29 23.11

Glycyl-tRNA Synthetase 37.06 20.61 10.53 31.80

GTP pyrophosphokinase 50.13 20.56 7.43 21.88

ATP-dependent protease La 46.21 17.33 8.34 28.11

DNA-primase 47.02 21.36 7.78 23.84

Histidy-tRNA Synthetase 37.68 20.77 8.94 32.61

GTP-Binding protein 49.45 19.23 10.16 21.15

Excinuclease ABC Subunit Bs 51.13 17.14 10.23 21.50

tRNA Modification GTpass 45.80 22.35 7.74 24.12

Tyrosine-TRNA ligase 48.79 18.60 10.87 21.74

PTS system-IIBC component 40.37 26.08 10.63 22.92

Hypothetical Protein Mccp 3340 51.59 16.22 7.44 24.76

Dihydro folate-foly poly

glutamate synthase

41.19 28.18 8.67 21.95

Lysine-tRNA ligase 40.60 22.40 7.60 29.40

Prolipoprotein diacylglyceryl

tranferase

38.40 24.52 10.08 27.00

Chromosome segregation

ATPase

58.20 16.09 5.67 20.04

Cell division protein FtsY 52.83 15.09 9.20 22.88

Signal recognition particle

protein

53.91 13.87 8.41 24.16

Parameters:

Window width: 17

Similarity threshold: 8

Number of states: 4

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Image coloured by rainbow N → C terminus

Model dimensions (Å): X:69.827 Y:47.914 Z:70.606 Figure 1: Schematic 3D structure of goat Phosphoglycerate_kinase-caprine protein

Discussion

CCPP diseases disease notifiable to the World Organization for Animal

Health (OIE) since it has a major impact on livestock production and a potential for

rapid spread across national borders. As a result, CCPP-infected countries are

excluded from international trade. At present, the disease causes vast problems in

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Africa with severe socio-economical consequences. The computed isoelectric

points (pI) for both CCPP will be useful for developing buffer system for

purification by isoelectric focusing method. The isoelectric point is of significance

in protein purification because it is the pH at which solubility is always minimal

and at which mobility in an electro focusing system is zero and therefore the point

at which the protein will accumulate (Fennema, 2008).

The extinction coefficient of a protein at 280 nm depends almost exclusively on the

number of aromatic residues, particularly tryptophan (Gill and Von-Hippel, 1989).

This indicates that the higher the EC value of the CCPP proteins, the higher the

number of aromatic residues (Gasteiger 2003; Munduganore et al., 2012). In

particular, hydrophobic amino acids can be involved in binding/recognition of

hydrophobic ligands such as lipids (Betts et al., 2003). All the CCPP proteins have

zero selenocystein and pyrrolysine which is interpret as stop codons (protein

cannot conclusively determine the identity of a residue) (Suchanek et al., 2005).

Many important biological processes such as cell signaling, transport of

membrane-impermeable molecules, cell–cell communication, cell recognition and

cell adhesion are mediated by membrane proteins (Jones, 2007). Although there

has been some recent progress in predicting the full 3-D structure of

transmembrane proteins (e.g. Yarov-Yarovoy et al., 2006), the most widely applied

prediction technique for these proteins is to determine the transmembrane

topology, i.e. the inside–outside location of the N and C termini relative to the

cytoplasm, along with the number and sequence locations of the membrane

spanning regions. This will facilitate the understanding of the structure and

function of CCPP proteins.

Determining the structure and function of a novel protein is a cornerstone of many

aspects of modern biology. The accuracy of protein structure prediction depends

critically on sequence similarity between the query and template as observed in the

present study. If a template is detected with >30% sequence identity to the query,

then usually most or all of the alignment will be accurate and the resulting relative

positions of structural elements in the model will be reliable (Kelley et al., 2015).

The practical applications of CCPP protein structure prediction include guiding the

development of functional hypotheses about hypothetical proteins, improving

phasing signals in crystallography and selecting sites for mutagenesis (Qian et al.,

2007; Rava and Hussain, 2007).

Conclusion

The physico-chemical properties, amino acid composition, and secondary

structure of CCPP proteins indicated physical, chemical and thermal stability of the

protein molecules. These indicated that the proteins are resistant to mutation and

can withstand wide range of temperature. Genetic data revealed from this study

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will bring new insights into epidemiological questions. Molecular typing has been

instrumental in determining the population structure and evolution of pathogens.

Since CCPP has both economical and nutritional consequences, efforts should be

intensified towards finding sustainable genomic solutions to these deadly diseases

which continue to ravage the livestock industry. New typing tool may help improve

the surveillance and control of the disease, as well as to trace new epidemics.

Analiza sekvence proteina zarazne pleuropneumonije koza

Ayuba Dauda, Abdulmojeed Yakubu, Ihe Ndu Dim, Deeve Sebastian Gwaza

Rezime Ukupno od dvadeset (20) proteina zarazne pleuropneumonije pluća goveda

(contagious bovine pleuropneumonia - CCPP) je preuzeto iz GenBank-a

(www.ncbi.nlm.nih.gov). Sekvence proteina korišćene su za ispitivanje

molekularnog identiteta različitih CCPP proteina. Fizičko-hemijske osobine CCPP

proteina su analizirane korišćenjem protparam alata. Isoelektrična tačka (pI),

molekularna masa (MW), koeficijent ekstinkcije (EK); indeks nestabilnosti (II),

alifatski indeks (AI) i veliki prosek hidropatičnosti (GRAVY). Studija je otkrila da

su pI CCPP proteina bili kiseli i bazni po svojoj prirodi. EC i II proteina CCPP

ukazuju na bolju stabilnost koja je indikacija otpornosti na mutaciju i toplotnu

stabilnost. GRAVY CCPP proteina je otkrio da su neki pozitivni, dok su neki

negativni. Pozitivna vrednost ukazuje na rastvorljivost (hidrofilni) u vodi, dok

negativni nije rastvorljiv (hidrofobni) u vodi. Sastav amino kiselina proteina

CCPP-a ukazuje na to da su bogati isoleucinom, leucinom i lizinom.

Trodimenzionalne strukture (3D) proteina CCPP su određene pomoću Phyre2

servera. Aminokislinske sekvence CCPP proteina su podvrgnute predviđanju

sekundarne strukture korišćenjem ExPASy’s SOPMA alata. Proteini su više

strukture alfa heliksa. Genetske informacije koje su rezultat ove studije mogu

doneti uvid u mutagenezu i farmakogenetiku.

Ključne reči: protein, pleuropneumonija koza, sekvenca

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Received 26 June 2017; accepted for publication 14 August 2017

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Schonbrun%20J%5Bauth%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/?term=Baker%20D%5Bauth%5D

distribution of moulds and mycotoxins in maize...

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